Antibodies against il-17br

ABSTRACT

The invention provides the antibody D9.2 and antibody molecules based on D9.2 which bind interleukin-17 receptor B. These may be useful in therapy, e.g. the treatment of asthma, ulcerative colitis or Crohn&#39;s disease.

FIELD OF THE INVENTION

The present invention relates to antibodies, including binding fragmentsthereof, directed to interleukin 17B receptor (IL-17BR).

BACKGROUND TO THE INVENTION

Asthma is a common chronic inflammatory disorder of the airways. Thenumber of sufferers has increased dramatically over recent decades andthe World Health Organisation estimates that in the region of 300million people worldwide suffer from asthma. Allergic asthma ischaracterised by uncontrollable airways hyperresponsiveness (AHR)induced by a variety of provocative stimuli and is associated withtype-2 inflammatory infiltrates into the lungs.

Inflammatory bowel disease (IBD) is a chronic inflammation affecting themucosal layer of the colon (also known as the large intestine), whichincludes two disease conditions: ulcerative colitis (UC) and Crohn'sdisease (CD). Conventional therapies for treatment of IBD involve eitherantibiotics or steroid-derived drugs or anti-TNF-α agents; however,these are not currently successful in inducing or maintaining clinicalremission in patients (Hanauer et al., 2008). UC is thought to be aTh2-mediated disease, with a representative mouse model showinginvolvement of type 2 cytokines in the development of gut inflammation(Heller et al., 2002)

The interleukin-17B receptor, variously known as IL-25R, IL-17BR,IL-17RB or IL-17RH1 was first identified in an expressed sequence tagdatabase by its homology to the IL-17A receptor (IL-17RA) (Tian et al.,2000). IL-17BR has subsequently been shown to bind both IL-17B and IL-25(Lee et al., 2001; Shi et al., 2000; Tian et al., 2000). IL-25 binds toIL-17BR with a stronger affinity (1.4 nM) than IL-17B (7.6 nM).

IL-25, a member of the IL-17 cytokine family (IL-17A, IL-17B, IL-17C,IL-17D and IL-17F—associated with type-1 inflammation), differsstrikingly from other IL-17 family members in that its productioninduces type-2 cytokine expression associated with splenomegaly,elevated serum levels of IgG1 and IgE and pathological changes in thelungs and digestive tract including eosinophilic infiltrates, increasedmucus secretion and epithelial cell hyperplasia (Fort et al., 2001; Leeet al., 2001; Moseley et al., 2003; Pan et al., 2001). Genetic ablationof IL-25 or the use of blocking anti-IL-25 antibodies have clearlydemonstrated the importance of IL-25 in protecting from helminthinfection (Fallon et al., 2006; Owyang et al., 2006), but also itscritical role in regulating responses characteristic of asthma(Ballantyne et al., 2007). It appears that IL-25 stimulates theseresponses through its ability to induce the release of type-2 cytokines,such as IL-13, initially from innate non-B/non-T (NBNT) cells (Fallon etal., 2006; Fort et al., 2001) and subsequently from the adaptive T cellresponse (Angkasekwinai et al., 2007; Wang et al., 2007).

il17br message has been identified in libraries from lung, brain,pancreas, kidney, thyroid and eosinophils (Lee et al., 2001; Shi et al.,2000). Expression in lung smooth muscle cells seems to beimmunologically regulated (Lajoie-Kadoch et al., 2006).

Consistent with a role in asthma, IL-25 mRNA or protein has beendetected from a number of cell types found in the lung includingalveolar macrophages, mast cells, eosinophils, and basophils (Wang etal., 2007). More recently, IL-25 production by allergen-stimulated humanand mouse lung epithelial cells has supported a potential role for IL-25modulating allergic pulmonary responses (Angkasekwinai et al., 2007). Inaddition, IL-25 has been reported to induce inflammatory cytokine andchemokine production from lung fibroblasts, and components ofextra-cellular matrix from airway smooth muscle cells. Furthermore,recent studies have indicated that transcripts for IL-25 and IL-17BR aresignificantly upregulated in biopsy tissue from asthmatic patients,associated with eosinophilic infiltration (Wang et al., 2007). Treatmentof OVA sensitised mice with a blocking monoclonal antibody directedagainst IL-25 results in a decreased AHR and lower IL-13 concentrationsin the bronchoalveolar lavage in response to OVA challenge andmethacholine administration.

Recently, Rickel et al. (J Immunol 181, 4299-4310 (2008)) used ablocking monoclonal antibody to human IL-17RA to prevent IL-25 activityin a primary human cell-based assay. This showed that IL-25 activityrequires both IL-17BR and IL-17RA. However, it has also been reportedthat IL-17A and IL-17F signal through a heteromeric complex containingIL-17RA and IL-17RC.

Rickel et al. also describe an antibody reactive with mouse IL-17BRwhich blocks IL-25-induced lung inflammation in a mouse model ofallergic asthma. To date, no antibodies reactive with human IL-17BR havebeen reported.

Consistent with a role in IBD, IL-25 production has been observed in anexperimental model of chronic colitis in mouse, in association with aswitch from a Th1 to a Th2 type response (Fichtner-Feigl et al., 2008)and high mRNA expression of IL-25 was found throughout thegastrointestinal tract in mice (Fort et al., 2001). Moreover the IL-25gene is located within a Crohn's disease susceptibility region onchromosome 14 in humans, although its potential association with thedisease remains to be investigated (Buning et al., 2003).

DISCLOSURE OF THE INVENTION

The present inventors have identified an antibody molecule which bindswith high affinity and specificity to IL-17BR.

Antibody molecules described herein may be useful in blocking IL-25bioactivity in vivo, and preventing airways inflammation, AHR, andinflammation of the colon.

In antigen challenged mice, administration of an antibody moleculedescribed herein is shown to reduce levels of IL-13 and IL-5, both ofwhich are critical cytokines in asthma regulation, and to reduce numbersof IL-13-producing cells in the lungs to levels and numbers similar tothose found in the absence of antigen challenge or in antigen challengedIL-17BR-deficient mice. Antibody molecules described herein may alsosignificantly reduce airways hyperreactivity. Furthermore, antibodymolecules described herein may reduce disease-related expansion ofgamma/delta T cells in vivo. This provides indication that antibodymolecules described herein inhibit two pathways known to be essential inthe development of asthma; IL-13 production and gamma/delta T cellresponsiveness.

The above described antigen challenged mice were challenged with OVA(ovalbumin) antigen in an experimental model of asthma. In micechallenged with OXA (oxazolone) antigen in an experimental model of IBD,administration of an antibody molecule described herein is shown toreduce the mortality rate and clinical signs of IBD, such as weight lossand the shortening of the colon that results from inflammation andhaemorrhage.

Antibody molecules described herein may cross-react with both mouse andhuman IL-17BR and may inhibit binding of human IL-25 to the humanIL-17BR. Thus, the antibody molecules may be used in mouse models toinvestigate its mechanisms of action in vivo. Furthermore, antibodymolecules described herein may block the biological action of theIL-25/IL-17BR complex in human cells. Antibody molecules describedherein therefore have potential utility for treatment of disease, suchas asthma.

An aspect of the invention provides an antibody molecule which bindsIL-17BR and which comprises an antibody VH domain comprising a VH CDR3with the amino acid sequence of SEQ ID NO: 7.

Preferably, an antibody molecule blocks IL-17BR binding to IL-25 andreduces or inhibits at least one of IL-25-mediated AHR; IL-13production; IL-25-mediated IL-5 production; IL-25-mediated IL-8production; and gamma/delta T cell expansion and infiltration.

An antibody molecule may comprise a VH domain which comprises a VH CDR3of SEQ ID NO: 7 together with a CDR1 of SEQ ID NO: 5 and a CDR2 of SEQID NO: 6.

A VH domain may be paired with a VL domain, for example a VL domain witha CDR1 of SEQ ID NO: 8, a CDR2 of SEQ ID NO: 9 and a CDR3 of SEQ ID NO:10. In some embodiments, a VH domain may be paired with a VL domain ofSEQ ID NO: 4.

In some embodiments, an antibody molecule may comprise a VH domain whichcomprises a VH CDR1 of SEQ ID NO:5, a VH CDR2 of SEQ ID NO:6 and a VHCDR3 of SEQ ID NO:7 and a VL domain which comprises a VL CDR1 of SEQ IDNO:8, a VL CDR2 of SEQ ID NO:9 and a VL CDR3 of SEQ ID NO:10.

A VH domain may further comprise human or non-human framework regions,for example the framework regions shown in SEQ ID NO: 2. In someembodiments, the antibody molecule may comprise the VH domain of SEQ IDNO: 2.

A VL domain may further comprise human or non-human framework regions,for example the framework region shown in SEQ ID NO: 4. In someembodiments, the antibody molecule may comprise the VL domain of SEQ IDNO: 4.

In some embodiments, the antibody molecule may comprise the VH domain ofSEQ ID NO: 2 and the VL domain of SEQ ID NO: 4.

Aspects of the invention also provide isolated nucleic acid encoding theantibody molecules described herein, vectors comprising the nucleic acidand methods of expressing the nucleic acid in a host cell to produceantibody molecules of the invention.

The invention further provides the use of antibody molecules of theinvention, for example in the form of a pharmaceutical composition, forthe treatment of disease, for example IL-25 mediated diseases such asallergy, asthma and colitis.

These and further aspects of the invention are described in furtherdetail below and with reference to the accompanying examples.

DESCRIPTION OF THE FIGURES

FIG. 1 shows that anti-IL-17BR clone D9.2 is cross-reactive for humanand murine IL-17BR binding by ELISA. Bars show binding of media (whitebars) or D9.2 antibody clone (black bars) to immobilised murineIL-17BR-Fc, human IL-17BR-Fc or human IgG control.

FIG. 2 shows D9.2 binds IL-17BR on transfected COS7 cells and on primarymouse cells. COS7 cells transfected with murine (a) or human (b) IL-17BRexpression vectors express IL-17BR that is recognised by D9.2. Cellswere incubated with 1 μg/ml D9.2 for 20 minutes and then washed. D9.2binding was detected by FACS after 20 minute incubation with anti-mouseIgG FITC at 0.5 μg/ml. (c) In vitro differentiated Th2 cells expressIL-17BR and are bound by D9.2. Expression is low or absent on Th1 cells.(d) After three consecutive daily intraperitoneal doses of 400 ng IL-25,a population of 1′-17BR-expressing non-B non-T (NBNT) cells appears inthe mysenteric lymph node of wild-type (d, e) but not IL-17BR knock-out(d, f) mice. This population is recognised by D9.2 and produces IL-13.

FIG. 3 shows that D9.2 binds IL-17BR but does not cross-react with mouseor human IL-17RA, IL-17RC, or IL-17RD. In brief, ELISA plates werecoated with IL-17R-family members; IL-17RA, IL-17BR, IL-17RC or IL-17RD,or IL-13Rα control, at 2 μg/ml, incubated overnight at 4° C., washed inPBS/0.05% tween and blocked in PBS/10% FCS at room temperature for 4hrs. Biotinylated D9.2 binding was detected using streptavidin-HRP andELISA development solution and measuring absorbance at 405 nm.

FIG. 4 shows that mouse monoclonal antibody D9.2, but not other mousemonoclonal anti-IL17BR antibodies (3b9, 2c1, 5h9, 7e1, 10c6, 10e6),inhibits the binding of human IL-25 to the human IL-25 receptor. Eachpurified mouse monoclonal antibody was diluted (×100) in hIL17Br/Fc (100ng/ml) to a final concentration of 1 μg/ml in a hIL17Br/Fc solution at100 ng/ml. In brief, [antibody×hIL17Br/Fc] mix was added to humanIL-25-coated plates, incubated 1 hour 30 mins and washed three times.Anti-hIgG (Fc)-HRP conjugate (Serotec) was added to the plate andincubated for 45 mins at RT, washed three times and developed with TMB.Reaction was stopped with 1 M HCL. Optical density was read at 450 nm,and the reagent blank reading subtracted from all readings.

FIG. 5 shows that D9.2 antibodies block NBNT cell and CD4+ (T and/orNKT) cell IL-13 production in vitro. (a) NBNT cell. In brief, mysentericlymph nodes were excised from naïve BALB/c mice and depleted of CD3+ andCD19+ cells. NBNT cells were plated on round bottomed 96-well plates at3×10⁵ cells/well and incubated for 72 hrs in RPMI 10% alone (MEDIA) orRPMI 10% FCS with 10 ng/ml IL-25 (IL-25). D9.2 was added to wells inserial dilution from a top concentration of 2 μg/ml and incubated for1.5 hrs before addition of 10 ng/ml IL-25 to the wells. Plates were thenincubated at 37° C. for 72 hrs before supernatants were tested for IL-13content by ELISA. (b) CD4+ (T and/or NKT) cell. Spleens were taken fromnaïve wild-type BALB/c mice and a single cell suspension prepared.Isolated CD4+ cells were cultured at 1×10⁶ cells/ml in 96-well plateseither in RPMI alone or in RPMI supplemented with 10 ng/ml IL-25 with orwithout D9.2 at 1 μg/ml. Cells were cultured for 72 hrs beforesupernatants were taken for analysis of IL-13 protein levels by ELISA.

FIG. 6 shows that D9.2 inhibits IL-25-induced production of KC (mouseIL-8) from a mouse renal carcinoma (RENCA) cell line in a dose-dependentmanner. IL-25 (100 ng/ml) was pre-incubated with varying concentrationsof D9.2 or IgG1 control antibody for 30-60 minutes at room temperatureprior to addition to cells. TNF-α (10 ng/ml) was added to RENCAcell-coated plates immediately prior to addition of the IL-25protein/D9.2 mix. Control samples without antibody were incubated with100 ng/ml of IL-25 and 10 ng/ml TNF-α (IL-25+TNF-α), or medium onlycontrol (Media), for 24 hrs before adding to the cells. Determination ofKC release by ELISA was performed at 24 hrs post stimulation.

FIG. 7 shows that D9.2 inhibits IL-25-induced production of IL-8 from ahuman renal carcinoma (TK-10) cell line in a dose-dependent manner.IL-25 (100 ng/ml) was pre-incubated with varying concentrations of D9.2or IgG1 control antibody for 30-60 minutes at room temperature. TNF-α(10 ng/ml) was added to TK-10 cell-coated plates immediately prior toaddition of the IL-25 protein/D9.2 mix. Control samples without antibodywere incubated with 100 ng/ml of IL-25 and 10 ng/ml TNF-α (IL-25+TNF-α),or medium only control (Media), for 24 hrs before adding to the cells.Determination of IL-8 release by ELISA was performed at 24 hrs poststimulation.

FIG. 8 shows that D9.2 blocks IL-25 responses in vivo. (a) Mediastinallymph node cells from ovalbumin (OVA) sensitised and challenged miceproduce IL-13 when restimulated with OVA in vitro. Administration ofD9.2 prior to OVA challenge reduces the IL-13 response to levelscomparable with those of IL-17BR KO mice. (b) Mediastinal lymph nodecells from OVA sensitised and challenged mice produce IL-5 whenrestimulated with OVA in vitro. Administration of D9.2 prior to OVAchallenge reduces the IL-5 response to levels comparable with those ofIL-17BR KO mice. (c) D9.2 treatment reduced the number ofIL-13-producing cells in the lung of OVA sensitised and challenged mice.Intracellular cytokine staining of IL-13 reveals a population ofIL-13-producing cells in OVA sensitised and challenged mice which isabsent in IL-17BR KO mice and is reduced in animals treated withanti-IL-17BR antibody D9.2 prior to OVA challenge. (d) Gamma-deltaT-cell numbers in the spleen of OVA sensitised and challenged mice arereduced in IL-17BR KO mice and in mice given D9.2 prior to OVAchallenge. (e) D9.2 treatment reduces AHR in OVA sensitised andchallenged mice.

FIG. 9 shows that D9.2 blocks IL-25 responses in vivo in a mouse modelof IBD. (a) Percentage survival of animals challenged with oxazolone islower than controls, receiving only ethanol. Administration of D9.2prior to sensitization and challenge reduces the mortality rate. (b)Clinical Scores are calculated based on weight loss in combination withthe general appearance and behaviour of each animal. D9.2 administrationresults in an improvement of the clinical score in comparison to micereceiving isotype antibody. (c) Colon length is reduced in IBD micecompared with controls. D9.2 administration protects against thisreduction in the IBD animals.

Sequences:

The antibody molecules of the present invention are described furtherherein with reference to the following sequence identification numbers:

SEQ ID NO:1 D9.2 VH encoding nucleotide sequenceSEQ ID NO:2 D9.2 VH amino acid sequenceSEQ ID NO:3 D9.2 VL encoding nucleotide sequenceSEQ ID NO:4 D9.2 VL amino acid sequenceSEQ ID NO:5 D9.2 VH CDR1 amino acid sequenceSEQ ID NO:6 D9.2 VH CDR2 amino acid sequenceSEQ ID NO:7 D9.2 VH CDR3 amino acid sequenceSEQ ID NO:8 D9.2 VL CDR1 amino acid sequenceSEQ ID NO:9 D9.2 VL CDR2 amino acid sequenceSEQ ID NO:10 D9.2 VL CDR3 amino acid sequence

Further sequences are set out in the accompanying sequence listing.

DETAILED DESCRIPTION OF THE INVENTION

This application is concerned with antigen-antibody type reactions.

In general, the heavy chain variable region (VH domain) of an antibodyplays a significant role in the binding of an antibody to an antigen.The CDR3 region of a VH domain has been found to be more diverse thanthe CDR1 and CDR2 regions, and thus in most antibodies providesspecificity for the target of the antibody. Thus antibody molecules ofthe invention are based around the VH CDR3 region of the D9.2 antibody.In some preferred embodiments, antibody molecules of the inventioncomprise all three CDRs of the VH regions of the D9.2 antibody.

The structure of an antibody molecule which comprises a CDR of theinvention will generally be of a heavy or light chain sequence of anantibody molecule or substantial portion thereof in which the CDR islocated at a location corresponding to the CDR of naturally occurring VHand VL antibody variable domains encoded by rearranged immunoglobulingenes. The structures and locations of immunoglobulin variable domainsmay be determined by reference to Kabat, E. A. et al, Sequences ofProteins of Immunological Interest. 4th Edition. US Department of Healthand Human Services. 1987, and updates thereof. A number of academic andcommercial on-line resources are available to query this database. Forexample, see Martin, A.C.R. Accessing the Kabat Antibody SequenceDatabase by Computer PROTEINS: Structure, Function and Genetics, 25(1996), 130-133 and the associated on-line resource, currently at theweb address of http://www.bioinf.org.uk/abs/simkab.html.

Generally, an antibody molecule comprises a VH domain which is pairedwith a VL domain to provide an antibody antigen binding domain, althoughin some embodiments, a VH domain alone may be used to bind antigen. Forexample, the D9.2 VH domain (SEQ ID NO: 2) may be paired with the D9.2VL domain (SEQ ID NO: 4), so that an antibody antigen binding site isformed which comprises both the D9.2 VH and VL domains. Alternatively,the D9.2 VH domain may be paired with a VL domain other than the D9.2 VLdomain.

Light-chain promiscuity is well established in the art, as discussedfurther herein.

An antibody molecule described herein may bind human IL-17BR and/ormouse IL-17BR. For example, an antibody molecule may bind human IL-17BRand show no binding or substantially no binding to mouse IL-17BR.Alternatively, an antibody molecule of the invention may bind mouseIL-17BR and show no binding or substantially no binding to humanIL-17BR.

Preferably, antibody molecules of the invention are cross reactive withboth human and mouse IL-17BR. For example, a cross reactive antibodymolecule binds both human IL-17BR and mouse IL-17BR.

An antibody molecule as described herein may bind IL-17BR with anaffinity which is substantially similar to that of D9.2, e.g. 90% to110% of the binding affinity of D9.2. An antibody molecule willgenerally be specific for IL-17BR. In other words, an antibody moleculemay bind IL-17BR but show no binding or substantially no binding toother members of the IL-17R family. Preferably, an antibody moleculespecific for IL-17BR binds IL-17BR but shows no binding or substantiallyno binding to IL-17RA, IL-17RC and/or IL-17RD.

Typically, specificity may be determined by means of a binding assaysuch as ELISA employing a panel of antigens.

Binding of an antibody molecule described herein with IL-17BR may beabolished by competition with recombinant IL-17BR.

Binding affinity and neutralisation potency of different antibodymolecules described herein can be compared under appropriate conditionsusing routine techniques.

Antibody molecules include any binding member or substance having anantibody antigen-binding site with the required specificity and/orbinding to IL-17BR. Examples of antibody molecules includeimmunoglobulin isotypes and their isotypic subclasses; antibodyfragments, such as Fab, Fab′, Fab′-SH, scFv, Fv, dAb and Fd; engineeredantibody molecules, such as Fab₂, Fab₃, diabodies, triabodies,tetrabodies and minibodies; and any other polypeptide comprising anantibody antigen-binding site, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an antigen binding domain, orequivalent, fused to another polypeptide are therefore included. Cloningand expression of chimeric antibodies are described in EP-A-0120694 andEP-A-0125023.

Examples of antibody molecules include (i) the Fab fragment consistingof VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VHand CH1 domains; (iii) the Fv fragment consisting of the VL and VHdomains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al.,Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolatedCDR regions; (vi) F(ab′)₂ fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883,1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and(ix) “diabodies”, multivalent or multispecific fragments constructed bygene fusion (WO94/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA90 6444-6448, 1993). Fv, scFv or diabody molecules may be stabilised bythe incorporation of disulphide bridges linking the VH and VL domains(Y. Reiter et al, Nature Biotech, 14, 1239-1245, 1996). Minibodiescomprising a scFv joined to a CH3 domain may also be made (S. Hu et al,Cancer Res., 56, 3055-3061, 1996). Antibody molecules and methods fortheir construction and use are described in Holliger & Hudson, NatureBiotechnology 23(9):1126-1136 (2005).

Where bispecific antibody molecules are to be used, these may beconventional bispecific antibodies, which can be manufactured in avariety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol.4, 446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas,or may be any of the bispecific antibody fragments mentioned above.Diabodies and scFv can be constructed without an Fc region, using onlyvariable domains, potentially reducing the effects of anti-idiotypicreaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against IL-217BR, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(J. B. B. Ridgeway et al, Protein Eng., 9, 616-621, 1996).

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodymolecules or chimeric molecules which retain the specificity of theoriginal antibody. Such techniques may involve introducing DNA encodingthe immunoglobulin variable region, or the complementarity determiningregions (CDRs), of an antibody to the constant regions, or constantregions plus framework regions, of a different immunoglobulin. See, forinstance, EPA-184187, GB 2188638A or EP-A-239400.

Preferably the CDR regions are grafted into a human framework region.The human framework region may be selected by a number of methods, e.g.by comparing the mouse framework region or mouse V region sequences withknown human framework or V region sequences and selecting a humanframework region which has the highest, or one of the highest degrees ofamino acid similarity or identity. Modifications to framework regions ofnative human sequences may be made in order to further optimize theresulting CDR-grafted antibodies.

Although antibody molecules comprising a pair of VH and VL domains arepreferred, single binding domains based on either VH or VL domainsequences may also be used. It is known that single immunoglobulindomains, especially VH domains, are capable of binding target antigensin a specific manner.

In the case of either of the single chain binding domains, these domainsmay be used to screen for complementary domains capable of forming atwo-domain antibody molecule able to bind IL-17BR, as discussed furtherherein below.

Antibody molecules may further comprise antibody constant regions orparts thereof. For example, a VL domain may be attached at itsC-terminal end to antibody light chain constant domains including humanCκ or Cλ chains, preferably Cλ chains. Similarly, an antibody moleculebased on a VH domain may be attached at its C-terminal end to all orpart of an immunoglobulin heavy chain derived from any antibody isotype,e.g. IgG, IgA, IgE and IgM and any of the isotype subclasses,particularly IgG1 and IgG4. IgG4 is preferred. Fc regions such as Δnaband Δnac as disclosed in WO99/58572 may be employed.

Framework regions of antibody molecules of the invention may alsoinclude glycosylation sequences that include one or more glycosylationsites. Depending upon the host cell in which the antibody is expressed,the pattern of glycosylation may vary. Thus nucleic acid constructs thatencode glycosylation sites may be modified to remove the site oralternatively such sites may be introduced into the protein. Forexample, N-glycosylation sites in eukaryotic proteins are characterizedby an amino acid triplet Asn-X-Y, wherein X is any amino acid except Proand Y is Ser or Thr. Appropriate substitutions, additions or deletionsto the nucleotide sequence encoding these triplets will result inprevention of attachment of carbohydrate residues at the Asn side chain.Alteration of a single nucleotide, chosen so that Asn is replaced by adifferent amino acid, for example, is sufficient to inactivate anN-glycosylation site. Known procedures for inactivating N-glycosylationsites in proteins include those described in U.S. Pat. No. 5,071,972 andEP 276,846.

The term “antigen-binding domain” describes the part of an antibodymolecule which comprises the area which specifically binds to and iscomplementary to part or all of an antigen. Where an antigen is large,an antibody may only bind to a particular part of the antigen, whichpart is termed an epitope. An antigen binding domain may be provided byone or more antibody variable domains (e.g. a so-called Fd antibodyfragment consisting of a VH domain). Preferably, an antigen bindingdomain comprises an antibody light chain variable region (VL) or atleast a substantial portion thereof and an antibody heavy chain variableregion (VH) or at least a substantial portion thereof.

A substantial portion of an immunoglobulin variable domain will compriseat least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of antibody moleculesmade by recombinant DNA techniques may result in the introduction of N-or C-terminal residues encoded by linkers introduced to facilitatecloning or other manipulation steps. Other manipulation steps includethe introduction of linkers to join variable domains of the invention tofurther protein sequences including immunoglobulin heavy chains, othervariable domains (for example in the production of diabodies) or proteinlabels as discussed in more detail below.

Antibody molecules and nucleic acid encoding antibody molecules willgenerally be isolated i.e. free or substantially free of material withwhich they are naturally associated such as other polypeptides ornucleic acids with which they are found in their natural environment, orthe environment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practised in vitro or invivo.

Antibody molecules and nucleic acid may be formulated with diluents oradjuvants and still for practical purposes be isolated—for example themolecules will normally be mixed with gelatin or other carriers if usedto coat microtitre plates for use in immunoassays, or will be mixed withpharmaceutically acceptable carriers or diluents when used in diagnosisor therapy. Antibody molecules may be glycosylated, either naturally orby systems of heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC85110503) cells), or they may be (for example if produced by expressionin a prokaryotic cell) unglycosylated.

In addition to antibody sequences, an antibody molecule as describedherein may comprise other amino acids, e.g. forming a peptide orpolypeptide, such as a folded domain, or to impart to the moleculeanother functional characteristic in addition to ability to bindantigen.

In some embodiments, antibody molecules may carry a detectable orfunctional label, or may be conjugated to a toxin or enzyme (e.g. via apeptidyl bond or linker).

A label can be any molecule that produces or can be induced to produce asignal, including but not limited to fluorescers, radiolabels, enzymes,chemiluminescers or photosensitizers. Thus, binding may be detectedand/or measured by detecting fluorescence or luminescence,radioactivity, enzyme activity or light absorbance.

Suitable labels include radiolabels such as ¹³¹I or ⁹⁹Tc, which may beattached to antibody molecules using conventional chemistry known in theart of antibody imaging. Labels also include enzyme labels such ashorseradish peroxidase, alkaline phosphatase, glucose-6-phosphatedehydrogenase (“G6PDH”), alpha-D-galactosidase, glucose oxydase, glucoseamylase, carbonic anhydrase and acetylcholinesterase. Labels includefluorescent labels or fluorescers, such as fluorescein and itsderivatives, fluorochrome, rhodamine compounds and derivatives and GFP(GFP for “Green Fluorescent Protein”). Labels further include chemicalmoieties such as biotin which may be detected via binding to a specificcognate detectable moiety, e.g. labelled avidin.

Where the additional feature is a polypeptide domain or label, theantibody molecule may be produced by recombinant techniques, i.e. by theexpression of nucleic acid encoding a fusion of the antibody moleculeand the further domain.

Antibody molecules reactive with IL-17BR may comprise variants of the VHand VL domains and CDRs set out herein. Variants may be obtained bymeans of methods of sequence alteration or mutation and screening.

An antibody molecule according to the invention may also be one whichcompetes for binding to IL-17BR with any antibody molecule which bothbinds IL-17BR and comprises a VH and/or VL domain disclosed herein, morepreferably an antibody molecule comprising the VH domain of SEQ ID NO: 2and the VL domain of SEQ ID NO: 4. Thus, a further aspect of the presentinvention provides an antibody molecule comprising a human antibodyantigen-binding site which competes with D9.2 for binding to IL-17BR.Competition between antibody molecules may be assayed easily in vitro,for example using ELISA and/or by tagging a specific reporter moleculeto one antibody molecule which can be detected in the presence of otheruntagged antibody molecule(s), to enable identification of antibodymolecule(s) which bind the same epitope or an overlapping epitope.

Various methods are available in the art for obtaining antibodymolecules against IL-17BR and which may compete with D9.2 for binding toIL-17BR.

Variants of the variable domain amino acid sequences disclosed hereinmay be employed, as discussed. Particular variants may include one ormore amino acid sequence alterations (addition, deletion, substitutionand/or insertion of an amino acid residue), may be less than about 20alterations, less than about 15 alterations, less than about 10alterations or less than about 5 alterations, 4, 3, 2 or 1. Alterationsmay be made in one or more framework regions and/or one or more CDRs.

A CDR amino acid sequence substantially as set out herein may be carriedas a CDR in a human variable domain or a substantial portion thereof.For example, VH CDR3 sequences substantially as set out herein may becarried as a VH CDR3 in a human heavy chain variable domain or asubstantial portion thereof.

Another aspect of the invention provides an antibody molecule whichbinds IL-17BR and which comprises an antibody VH domain comprising a VHCDR3 substantially as set out in SEQ ID NO:7.

An antibody molecule may comprise a VH domain which comprises a VH CDR1,CDR2 and CDR3 substantially as set out in SEQ ID NOS: 5, 6 and 7,respectively.

A VH domain may be paired with a VL domain, for example a VL domain witha CDR1, CDR2 and CDR3 substantially as set out in SEQ ID NOS: 8, 9 and10, respectively.

In some embodiments, an antibody molecule may comprise a VH domain whichcomprises a VH CDR1, CDR2 and CDR3 substantially as set out in SEQ IDNOS: 5, 6 and 7, respectively; and, a VL domain with a CDR1, CDR2 andCDR3 substantially as set out in SEQ ID NOS: 8, 9 and 10, respectively.

The VH and VL domains may have human or non-human framework regions, forexample framework regions substantially as set out in the frameworkregions of SEQ ID NO: 2 and SEQ ID NO:4, respectively.

In some embodiments, the antibody molecule may comprise the VH and VLdomain sequences substantially as set out in SEQ ID NO: 2 and SEQ IDNO:4, respectively.

By “substantially as set out” it is meant that the relevant CDR or VH orVL domain of the invention will be either identical or highly similar tothe specified regions of which the sequence is set out herein. By“highly similar” it is contemplated that from 1 to 5, preferably from 1to 4 such as 1 to 3 or 1 or 2, or 3 or 4, amino acid substitutions maybe made in the CDR and/or VH or VL domain.

Sequence variants of antibody molecules may be generated by carrying outrandom mutagenesis of one or both of the D9.2 VH and/or VL genes togenerate mutations within the entire variable domain. Such a techniqueis described by Gram et al (1992, Proc. Natl. Acad. Sci., USA,89:3576-3580), who used error-prone PCR.

Another method which may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al, (1994,Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J.Mol. Biol. 263:551-567).

All the above described techniques are known as such in the art and inthemselves do not form part of the present invention. The skilled personwill be able to use such techniques to provide antibody molecules asdescribed herein using routine methodology in the art.

Accordingly, another aspect of the invention provides a method forobtaining an antibody molecule against IL-17BR which comprises:

-   -   providing a starting nucleic acid encoding a antibody molecule        that has one or more (i.e. one, two, three, four, five or all        six) of the CDR sequences of SEQ ID NO:2 or SEQ ID NO:4;    -   modifying said nucleic acid to alter the CDR sequence or        sequences;    -   expressing said modified antibody molecule; and    -   testing said modified antibody molecule for binding against        IL-17BR.

Preferably the modification will be performed on a plurality of startingnucleic acid molecules to provide a repertoire of modified sequenceshaving a diversity of binding affinities.

The starting nucleic acid preferably comprises all three heavy chainCDRs of SEQ ID NO: 2, either in the form of SEQ ID NO:2 itself orcarried in another framework sequence.

The modifications may be directed at a single CDR, e.g. the CDR3, or themodifications may be directed to two or three CDR regionssimultaneously.

Variable domains employed in the invention may be obtained from anygerm-line or rearranged human variable domain, or may be a syntheticvariable domain based on consensus sequences of known human variabledomains. A CDR sequence as described herein (e.g. CDR3) may beintroduced into a repertoire of variable domains lacking a CDR(particularly CDR3), using recombinant DNA technology.

For example, Marks et al (Bio/Technology, 1992, 10:779-783) describemethods of producing repertoires of antibody variable domains in whichconsensus primers directed at or adjacent to the 5′ end of the variabledomain area are used in conjunction with consensus primers to the thirdframework region of human VH genes to provide a repertoire of VHvariable domains lacking a CDR3. Marks et al further describe how thisrepertoire may be combined with a CDR3 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide antibody molecules as described herein. Therepertoire may then be displayed in a suitable host system such as thephage display system of WO92/01047 so that suitable antibody moleculesmay be selected. A repertoire may consist of from anything from 10⁴individual members upwards, for example from 10⁶ to 10⁸ or 10¹⁰ members.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature, 1994, 370:389-391), who describes the technique inrelation to a β-lactamase gene but observes that the approach may beused for the generation of antibodies.

A further aspect of the invention thus provides a method of preparing anantibody molecule specific for IL-17BR, which method comprises:

-   -   (a) providing a starting repertoire of nucleic acids encoding a        VH domain which either include a CDR3 to be replaced or lack a        CDR3 encoding region;    -   (b) combining said repertoire with a donor nucleic acid encoding        an amino acid sequence substantially as set out in SEQ ID NO:7        such that said donor nucleic acid is inserted into the CDR3        region in the repertoire, so as to provide a product repertoire        of nucleic acids encoding a VH domain;    -   (c) expressing the nucleic acids of said product repertoire;    -   (d) selecting a antibody molecule specific for a IL-17BR; and    -   (e) recovering said antibody molecule or nucleic acid encoding        it.

The product repertoire may be co-expressed, from the same vector ordifferent vector, with a VL domain. The VL domain may be a VL domaindescribed herein e.g. the VL domain of SEQ ID NO: 4, or may be one ormore different VL domains, as described below in relation to chainshuffling.

An analogous method may be employed in which a VL CDR3 substantially asset out in SEQ ID NO: 10 is combined with a repertoire of nucleic acidsencoding a VL domain which either include a CDR3 to be replaced or lacka CDR3 encoding region. As with the method above, the VL productrepertoire may be co-expressed, from the same vector or differentvector, with a VH domain. The VH domain may be a VH domain describedherein i.e. the VH domain of SEQ ID NO: 2 or may be one or moredifferent VH domains, as described below in relation to chain shuffling.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains which are then screened for an antibodymolecule or antibody molecules specific for IL-17BR.

Antibody molecules obtained in this manner form a further aspect of theinvention.

Another aspect of the invention provides a method for obtaining anantibody antigen-binding domain for IL-17BR, the method comprisingcombining a VH domain of an antibody molecule described herein(including variants as discussed above) with one or more VL domains, andtesting the VH/VL combination or combinations for antibody-antigenbinding domain for IL-17BR.

Said VL domain may have an amino acid sequence which is substantially asset out herein. For example, the VL domain may be substantially as setout in SEQ ID NO: 4.

An analogous method may be employed in which one or more sequencevariants of a VL domain disclosed herein are combined with one or moreVH domains.

This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed inWO92/01047 in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain antibody molecule isselected in accordance with phage display techniques such as thosedescribed in that reference.

Another aspect of the present invention provides a method for selectionof an antibody molecule for IL-17BR, the method comprising:

-   -   (a) providing an antibody VH domain comprising a VH CDR3 with        the amino acid sequence of SEQ ID NO. 7;    -   (b) combining said VH domain with a plurality of antibody VL        domains to provide antibody molecules;    -   (c) screening said antibody molecules for binding to IL-17BR;        and    -   (d) selecting an antibody molecule which binds IL-17BR.

In such a method, the VH and VL domains may be provided in the form ofproteins expressed by recombinant DNA, particularly by a phage orphagemid DNA.

The plurality of VL domains may be anything from 10⁴ individual domainsupwards, for example from 10⁶ to 10⁸ or 10¹⁰ domains.

IL-17BR, also referred to in the art as IL-25R, IL-17RB or IL-17RH1, isavailable from commercial sources (e.g. R&D Systems, MN, USA) as anFc-fusion protein, or may be cloned or synthesised by reference to thesequences of IL-17BR available in the art.

Murine IL-17BR (GeneID: Nucleic acid: NM_(—)019583.3 GI:142368701;NP_(—)062529.2 GI:83025064) is described by Tian et al., 2000 (Ref. 11below). Human IL-17BR (GeneID: 55540, Nucleic acid: NM_(—)018725.3GI:112382255; Protein NP_(—)061195.2 GI:27477074) is described by Shi,Y., et al., 2000 (Ref. 10 below).

For production of antibodies or use in immunoassays, fragments ofrecombinant IL-17BR may be used, particularly those containing theextracellular domain.

In further aspects, the invention provides an isolated nucleic acidwhich comprises a nucleotide sequence encoding an antibody molecule, aVH domain, or a VL domain as described above, for example a VH or VLdomain of SEQ ID NOS: 2 and 4 respectively, and methods of preparing anantibody molecule, a VH domain, or a VL domain as described above, whichcomprise expressing said nucleic acid under conditions to bring aboutproduction of said antibody molecule, VH domain, or VL domain, andrecovering it.

Another aspect of the present invention provides nucleic acid, generallyisolated, encoding a VH CDR or VL CDR sequence disclosed herein,especially a VH CDR selected from SEQ ID NOs: 5, 6 and 7, a VL CDRselected from SEQ ID NOs: 8, 9 and 10, most preferably D9.2 VH CDR3 (SEQID NO. 7).

The nucleic acids of the invention may comprise the sequences, orrelevant portions thereof (e.g. CDR-encoding regions) of SEQ ID NO:1 orSEQ ID NO:3, or variants of these sequences modified by, for example,site-directed mutagenesis to encode other VH and VL domains of theinvention. However, codon usage may be varied, e.g. to optimizeexpression of the sequence in a desired host cell.

Another aspect of the present invention provides an isolated nucleicacid encoding an antibody molecule of the present invention. Nucleicacid includes DNA and RNA. In a preferred aspect, the present inventionprovides a nucleic acid which encodes a CDR or a VH or VL domain of theinvention as defined above.

Nucleic acid according to the present invention may comprise DNA or RNAand may be wholly or partially synthetic. Reference to a nucleotidesequence as set out herein encompasses a DNA molecule with the specifiedsequence, and encompasses a RNA molecule with the specified sequence inwhich U is substituted for T, unless context requires otherwise.

Aspects of the present invention also provide vectors, for example inthe form of plasmids, viruses, e.g. phage, or phagemid, cosmids,transcription or expression cassettes which comprise at least onenucleic acid as above.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Vectors also includeviral vectors capable of infecting human cells in vivo, e.g. adenoviral,retroviral or adeno-associated virus vectors. Such vectors may be usefulfor expression of an antibody molecule of the invention in the cells ofa human or animal subject, to provide for production and delivery of theantibody molecule to said subject.

A nucleic acid sequence encoding an antibody molecule of the inventionwill in one aspect be operably linked to a promoter to effect expressionof the antibody molecule in a host cell. The sequence may include at its5′ end a leader sequence to facilitate expression and/or secretion ofthe antibody molecule in and/or from a host cell. Numerous suitableleader sequences are known as such in the art and may be selected by aperson of ordinary skill in the art taking account of the host cell.

Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Current Protocols inMolecular Biology, Second Edition, Ausubel et al. eds. John Wiley &Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference.

Another aspect provides a host cell transformed with a nucleic acid(e.g. a nucleic acid sequence in the form of a vector) of the invention.

Nucleic acid may be integrated into the genome (e.g. chromosome) of thehost cell. Integration may be promoted by inclusion of sequences whichpromote recombination with the genome, in accordance with standardtechniques.

Another aspect provides a method of production of an antibody moleculeas described herein, the method including causing expression fromencoding nucleic acid. Such a method may comprise culturing host cellsunder conditions for production of said antibody molecule.

Following production by expression, a VH or VL domain, or antibodymolecule may be isolated and/or purified using any suitable technique,then used as appropriate. A method of production may comprise a step ofisolation and/or purification of the product.

Following purification of the product the antibody molecule may bemodified by physical or chemical means, for example to introduceprotective groups that alter, e.g. increase, the stability or biologicalhalf-life of the protein. For example, PEGylation of proteins to achievesuch effects is known as such in the art and antibody molecules of theinvention may be in PEGylated form.

A method of production may comprise formulating the product into acomposition including at least one additional component, such as apharmaceutically acceptable excipient.

The present invention also provides a recombinant host cell whichcomprises one or nucleic acids or vectors as above.

Systems for cloning and expression of an antibody molecule in a varietyof different host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells and manyothers. A common, preferred bacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Plückthun, A. Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of an antibody molecule, see for recentreviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4:573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

The data set out herein shows for the first time that antibodies againstIL-17BR are effective in preventing or reducing airwayhyperresponsiveness in vivo, a key symptom of asthma.

Another aspect of the invention provides a method of preventing orreducing airway hyperresponsiveness in a subject (e.g. a human) in needthereof which comprises administering to the subject an antibodymolecule which binds IL-17BR, for example an antibody molecule describedabove. Another aspect of the invention provides a method of preventing,reducing or treating asthma or other IL-25 mediated condition in asubject in need thereof which comprises administering to the subject anantibody molecule that binds IL-17BR. Other IL-25 mediated conditionsinclude allergy and colitis, which includes ulcerative colitis andCrohn's disease. Asthma includes allergic asthma.

Accordingly, another aspect of the invention provides a method ofpreventing or reducing inflammation of the colon in a subject (e.g. ahuman) in need thereof, which comprises administering to the subject anantibody molecule that binds IL-17BR, for example an antibody moleculedescribed above. Another aspect of the invention provides a method ofpreventing, reducing or treating IBD, which comprises administering tothe subject an antibody molecule that binds IL-17BR. IL-25 mediatedconditions include ulcerative colitis and Crohn's disease. Other IL-25mediated conditions include colitis (inflammation of the colon),including chronic colitis.

The above methods may be practiced with antibody molecules (includingcompositions thereof) as described above, which are useful in binding toIL-17BR and antagonising the effects of IL-17BR/IL-25 binding, withtherapeutic potential in various diseases and disorders in which IL-17BRplays a role. The methods may also be practiced with other antibodymolecules (including compositions thereof) which bind IL-17BR andantagonise the effects of IL-17BR/IL-25 binding, which may be obtainedas described below in the accompanying examples.

Antibody molecules (including compositions thereof) described, above maybe used in a method of treatment (including prophylactic treatment) ordiagnosis in human or animal subject. Such a method of treatment ordiagnosis (which may include prophylactic treatment) may compriseadministering to said subject an effective amount of an antibodymolecule of the invention. Exemplary diseases and disorders arediscussed further below.

Also provided is the use of an antibody molecule (including acomposition thereof) described herein in the manufacture of a medicamentfor administration, to a human or animal subject.

Clinical indications in which an anti-IL-17BR antibody molecule may beused to provide therapeutic benefit include any condition in whichIL-17BR/IL-25 binding has pathological consequences. Thus in general,the antibody molecule described herein may be used in the treatment ofany IL-25 mediated condition, for example associated with an unwantedTh2 response or type-2 responses. In some embodiments, the antibodymolecule of the invention may be used for the treatment of allergy andasthma, particularly asthma. In some embodiments, the antibody moleculeof the invention may be used for the treatment of IBD, particularly thetreatment of UC and/or CD.

Anti-IL-17BR treatment may be given by injection (e.g. intravenously) orby local delivery methods. Anti-IL-17BR may be delivered bygene-mediated technologies. Alternative formulation strategies mayprovide preparations suitable for oral or suppository route. The routeof administration may be determined by the physicochemicalcharacteristics of the treatment, by special considerations for thedisease, to optimise efficacy or to minimise side-effects.

The compositions provided may be administered to individuals.Administration is preferably in a “therapeutically effective amount”,this being sufficient to show benefit to a patient. Such benefit may beat least amelioration of at least one symptom. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors. Appropriate doses ofantibody are well known in the art; see Ledermann J. A. et al. (1991)Int. J. Cancer 47: 659-664; Bagshawe K. D. et al. (1991) Antibody,Immunoconjugates and Radiopharmaceuticals 4: 915-922.

The precise dose will depend upon a number of factors, including whetherthe antibody is for diagnosis or for treatment, the size and location ofthe area to be treated, the precise nature of the antibody (e.g. wholeantibody, fragment or diabody), and the nature of any detectable labelor other molecule attached to the antibody. A typical antibody dose willbe in the range 0.5 mg-1.0 g, and this may be administered intravenouslyas a bolus or as an infusion over several hours as appropriate toachieve the required dose. Other modes of administration includeintravenous infusion over several hours, to achieve a similar totalcumulative dose. This is a dose for a single treatment of an adultpatient, which may be proportionally adjusted for children and infants,and also adjusted for other antibody formats in proportion to molecularweight. Treatments may be repeated at daily, twice-weekly, weekly ormonthly intervals, at the discretion of the physician.

A further mode of administration employs precoating of, or otherwiseincorporation into, indwelling devices, for which the optimal amount ofantibody will be determined by means of appropriate experiments.

An antibody molecule in some embodiments may be a monomeric fragment,such as F(ab) or scFv. Such antibody fragments may have the advantage ofa relatively short half life and less risk of platelet activation, whichmay be caused by receptor clustering. Clustering which gives rise toplatelet activation could be either of IL-17BR molecules or of IL-17BRwith FcγRII molecules, for instance.

If a whole antibody, is used, it is preferably in a form that is unableto activate and/or destroy platelets. The IgG4 isotype or alternatively“designer” isotypes derived from the IgG1 backbone (novel Fc geneconstructs WO99/58572, Clark, Armour, Williamson) are preferred choices.Smaller antibody fragments may be used, such as F(ab′)₂. In addition,whole antibodies or fragments (e.g. F(ab′)₂ or diabodies) with dualepitope specificity (e.g. for the epitopes recognised by scFv D9.2) maybe used. Although such an embodiment may promote receptor clustering, ahigh association rate to individual receptors may rule out this problem.

Antibody molecules described herein will usually be administered in theform of a pharmaceutical composition, which may comprise at least onecomponent in addition to the antibody molecule.

Thus pharmaceutical compositions according to the present invention, andfor use in accordance with the present invention, may comprise, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. intravenous.

Therapeutic formulations of the antibody molecule may be prepared forstorage by mixing the antibody molecule having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers (see e.g. “Remington: The Science and Practice of Pharmacy”,20th Edition, 2000, pub. Lippincott, Williams & Wilkins.), in the formof lyophilized powder or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

For the antibody molecule to be used for in vivo administration it mustbe sterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The antibody molecule ordinarily will be stored inlyophilized form or in solution.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

An antibody molecule of the invention may be administered alone or incombination with other treatments, either simultaneously or sequentiallydependent upon the condition to be treated. Other treatments may includethe administration of suitable doses of pain relief drugs such asnon-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol,ibuprofen or ketoprofen) or opiates such as morphine; the administrationof anti-emetics; or the administration of at least one other compoundactive against asthma, generally a bronchodilating agent which producesairway relaxation or enhances mucus clearance, e.g. a beta-agonist (e.g.salbutamol, salmeterol), disodium cromoglycate, steroids or an inhibitorof PDE_(IV).

Another aspect of the invention provides a method comprising causing orallowing binding of an antibody molecule as provided herein to IL-17BR.As noted, such binding may take place in vivo, e.g. followingadministration of an antibody molecule, or nucleic acid encoding anantibody molecule, or it may take place in vitro, for example in ELISA,Western blotting, immunocytochemistry, immuno-precipitation or affinitychromatography.

The amount of binding of antibody molecule to IL-17BR may be determined.Quantitation may be related to the amount of the antigen in a testsample, which may be of diagnostic interest.

The reactivities of antibody molecules on a sample may be determined byany appropriate means. Radioimmunoassay (RIA) is one possibility.Radioactive labelled antigen is mixed with unlabelled antigen (the testsample) and allowed to bind to the antibody molecule. Bound antigen isphysically separated from unbound antigen and the amount of radioactiveantigen bound to the antibody determined. The more antigen there is inthe test sample the less radioactive antigen will bind to the antibodymolecule. A competitive binding assay may also be used withnon-radioactive antigen, using antigen or an analogue linked to areporter molecule. The reporter molecule may be a fluorochrome, phosphoror laser dye with spectrally isolated absorption or emissioncharacteristics. Suitable fluorochromes include fluorescein, rhodamine,phycoerythrin and Texas Red. Suitable chromogenic dyes includediaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

The signals generated by individual antibody-reporter conjugates may beused to derive quantifiable absolute or relative data of the relevantantibody binding in samples (normal and test).

The present invention also provides the use of an antibody molecule asabove for measuring antigen levels in a competition assay, that is tosay a method of measuring the level of antigen in a sample by employingan antibody molecule as provided herein in a competition assay. This maybe where the physical separation of bound from unbound antigen is notrequired. Linking a reporter molecule to the antibody molecule so that aphysical or optical change occurs on binding is one possibility. Thereporter molecule may directly or indirectly generate detectable, andpreferably measurable, signals. The linkage of reporter molecules may bedirectly or indirectly, covalently, e.g. via a peptide bond ornon-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

The present invention also provides for measuring levels of antigendirectly, by employing an antibody molecule as described herein forexample in a biosensor system.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

The present invention further extends to an antibody molecule whichcompetes for binding to IL-17BR with any antibody molecule which bothbinds the antigen and comprises a VH and/or VL domain including a CDRwith amino acid substantially as set out herein or a VH and/or VL domainwith amino acid sequence substantially as set out herein. Competitionbetween antibody molecules may be assayed easily in vitro, for exampleby tagging a specific reporter molecule to one antibody molecule whichcan be detected in the presence of other untagged antibody molecule(s),to enable identification of antibody molecules which bind the sameepitope or an overlapping epitope. Competition may be determined forexample using ELISA or flow cytometry.

A competition reaction may be used to select one or more antibodymolecules such as derivatives of D9.2, which may have one or moreadditional or improved properties. This is analogous to the selectionmethod for D9.2 in accordance with the invention, except that IL-17BR isnot eluted from its mini-ligand but from an antibody molecule. This maybe important as it should yield a greater proportion of daughterantibody molecules which directly compete with the parent. Indeed suchdaughter antibody molecules as are selected may have a greater affinityfor the antigen than the parent (allowing for enhancements in aviditywhich may result from the display of more than one antibody molecule perphage). Current methods of selecting for “daughter” phage antibodymolecules of improved affinity include:

-   -   using concentrations of (labelled) target antigen lower than the        dissociation constant of the original parent antibody;    -   using excess unlabelled target antigen as a competitor as        demonstrated in Hawkins et al. (1992). However, they do not        necessarily specify that the “improved” antibody must        displace/occupy the same epitope as the parent. Incorporating        the elution step should yield a higher proportion of daughter        antibody molecules which do displace the parent. Daughter        antibody molecules selected in this way may bind a very similar        epitope to the parent antibody molecule, but with a greater        affinity.

In testing for competition a peptide fragment of IL-17BR may beemployed, especially a peptide including an epitope of interest. Apeptide having the epitope sequence plus one or more amino acids ateither end may be used. Such a peptide may be said to “consistessentially” of the specified sequence. Antibody molecules according tothe present invention may be such that their binding for IL-17BR isinhibited by a peptide with or including the sequence given. In testingfor this, a peptide with either sequence plus one or more amino acidsmay be used.

Antibody molecules which bind a specific peptide may be isolated forexample from a phage display library by panning with the peptide(s).

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.All documents mentioned in this specification are incorporated herein byreference in their entirety.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures described above.

EXAMPLES

Materials and Methods

Screening Supernatants by ELISA

96-well immunoplates (Nunc) were coated with 50 μl/well murine or humanIL-17Br-Fc fusion protein at 1 μg/ml in 0.1 M NaHCO₃ overnight at 4° C.or for 3 hrs at room temperature. The next day, wells were washed 5times with PBS 0.05% tween and then blocked in PBS 10% FCS for 4 hrs atroom temperature. For the same 4 hour period, supernatants fromhybridoma cell culture were incubated with 50 μg/ml hIgG. This was toblock any antibodies in the supernatants that had been raised againstthe Fc portion of the fusion protein. Supernatants that did not receivethis treatment were also included in the ELISA to give an indication ofthe amount of anti-Fc antibody in each sample.

Following the 4 hour blocking step, immunoplates were washed 5 times inPBS 0.05% tween and supernatants were added neat, at 50 μl/well.Supernatants were left on the plate overnight at 4° C. or for 3 hrs atroom temperature before the wells were washed 5 times in PBS 0.05%tween. 50 μl of 0.5 μg/ml anti-mouse immunoglobulins-HRP (DAKO) in PBS10% FCS was added to each well and left for one hour at room temperaturebefore a final 8 washes in PBS 0.05% tween were performed. Boundantibody was detected with an ELISA development solution and the A405recorded on a Tecan immunoplate reader.

To distinguish antibodies raised against the Fc portion of the fusionprotein from those directed against IL-17BR, control plates were coatedwith hIgG and supernatants were added following the protocol above.Samples that gave a high A405 on hIgG coated plates were not consideredfor further study.

Flow Cytometry Screening of Transfected COS7 Cells

cDNAs for the murine and human IL-17BR genes were separately cloned intothe pME18S expression vector and termed mIL17BR-pME18S andhIL17BR-pME18S respectively.

2×10⁶ COS7 cells in DMEM 10% FCS were plated onto a 10 cm dish andincubated overnight at 37° C. The following day, 4 μg mIL17BR-pME18S orhIL17BR-pME18S was mixed with 10 μl lipofectamine in 500 μl serum freeOptimem and incubated at room temperature for 30 minutes before beingadded onto the plated cells. Cells were then incubated at 37° C. for 6hrs before adding fresh media. Cells were harvested for FACS analysis 48hrs post-transfection.

For FACS analysis, transfected or non-transfected cells were incubatedwith candidate anti-IL17BR antibodies at varying concentrations in PBS2% FCS for 30 minutes. Cells were then washed before incubation withanti-mouse IgG FITC (BD Pharmingen) at 2 μg/ml in PBS 2% FCS for afurther 30 minutes. Finally, cells were washed twice in PBS 2% FCS andanalysed for IL-17Br expression on a Becton Dickinson FACScaliburmachine.

D9.2 Cross-Reactivity

ELISA plates were coated with IL-17R-family members; IL-17RA, IL-17BR,IL-17RC, or IL-17RD, or IL-13Rα control (R&D Systems) at 2 μg/mlovernight at 4° C. before washing in PBS/0.05% tween and blocking inPBS/10% FCS at room temperature for 4 hrs. Biotinylated D9.2 was addedat 1 μg/ml in PBS/10% FCS and incubated overnight at 4° C. Plates werethen washed before streptavidin-HRP was added and incubated for 1 hr atroom temperature. Plates were then washed a final time before addingELISA development solution and measuring the absorbance at 405 nm.

Mouse Monoclonal Antibody—Human IL-17BR Binding Assay

Human IL-25 (hIL17e)(R&D sys) was coated onto a Nunc Maxisorp microwellplate at 0.5 μg/ml and incubated for 1 hour 30 mins at room temperature.The plate was washed three times and then blocked with Tris/1% BSA for 1hour. hIL17Br/Fc chimeric (R&D sys) was diluted to 100 ng/ml. Eachpurified mouse monoclonal antibody was diluted (×100) in hIL17Br/Fc (100ng/ml) to a final concentration of 1 μg/ml in a hIL17Br/Fc solution at100 ng/ml. The [antibody×hIL17Br/Fc] mix was incubated 1 hour 30 mins atroom temperature. The [antibody×hIL17Br/Fc] mix was added to thehIL17e-coated plate and incubated 1 hour 30 mins before being washedthree times. Anti-hIgG (Fc)-HRP conjugate (Serotec) was added to theplate and incubated for 45 mins at RT before being washed three timesand developed with TMB. Reaction was stopped with 1 M HCL. Opticaldensity was read at 450 nm, and the reagent blank reading subtractedfrom all readings.

IL-17BR/IL25 Inhibition Assay

Mysenteric lymph nodes were removed from naïve BALB/c mice and passedthrough 70 μm cell-strainers to achieve a single-cell suspension. Cellswere washed in PBS 2% FCS and then T-cells and B-cells were depleted.This was achieved by incubating cells with biotinylated anti-CD19 andanti-CD3 antibodies at 5 μg/ml, on ice, for 30 minutes and thenincubating with anti-biotin Dynabeads (Invitrogen) at a concentration of4 beads per cell for 20 minutes at 4° C. The mixture was then washedbefore being passed over a magnet to separate labelled T- and B-cellsfrom the unlabelled non-B non-T (NBNT) cell fraction. Purity of the NBNTfraction was tested by FACS, staining for B220, CD4 and CD8.

NBNT cells were then plated on round bottomed 96-well plates at 3×10⁵cells/well and incubated for 72 hrs in RPMI 10% alone or RPMI 10% FCSwith 10 ng/ml IL-25. Candidate IL-17BR blocking antibodies were added towells in serial dilution from a top concentration of 2 μg/ml andincubated for 1.5 hrs before addition of 10 ng/ml IL-25 to the wells.Plates were then incubated at 37° C. for 72 hrs before supernatants wereharvested and tested for IL-13 protein content by Quantikine ELISA (R&Dsystems).

For CD4+ (T and/or NKT) cells, spleens were taken from naïve wild-typeBALB/c mice and a single cell suspension prepared. Red blood cell lysiswas performed before washing cells in MACS buffer (Miltenyi Biotec).CD4+ cell isolation was carried out by positive selection using CD4MicroBeads (Miltenyi Biotec) according to manufacturers instructions.CD4+ cells were then cultured at 1×10⁶ cells/ml in 96-well plates eitherin RPMI alone or in RPMI supplemented with 10 ng/ml IL-25 with orwithout D9.2 at 1 μg/ml. Cells were cultured for 72 hrs and thensupernatants taken for analysis of IL-13 protein levels by QuantikineELISA (R&D systems).

Renal Carcinoma Cell Line Bioassays

Human TK-10 renal carcinoma cells were obtained from the National CancerInstitute (NCI). RENCA cells were obtained from Cell Biology services,Centocor R&D. Both cell lines were maintained in DMEM growth medium with10% FCS at 37° C., 5% CO₂ in a humidified atmosphere. Cells were platedin 96-well flat bottom tissue culture treated plates at a density of2.5×10⁴ cells/well in a total volume of 100 μl complete growth medium.Following overnight incubation, the cells were washed with 1×PBS andthen incubated with 100 ng/ml of IL-25 and 10 ng/ml TNF-α in OptiMEMreduced serum medium, or medium only control, for 24 hr. Cellsupernatant was collected 20-24 hrs post IL-25 stimulation and stored at−20° C. for subsequent analysis of soluble KC/IL-8 release using a mouseQuantikine ELISA for KC or human Quanitkine ELISA for IL-8 (R&Dsystems).

D9.2 or IgG1 control were tested for ability to prevent IL-25-mediatedIL-8 (KC) release. For inhibition experiments, a constant amount ofIL-25 (100 ng/ml) was pre-incubated with varying concentrations of D9.2or anti-c-myc mouse IgG1 (clone 9E10.2) control antibody for 30-60minutes at room temperature prior to addition to the respective cells.TNF-α (10 ng/ml) was added to the cells immediately prior to addition ofthe IL-25 protein/D9.2. Determination of IL-8 (KC) release was performedat 24 hr post stimulation as described above.

Mice

BALB/c mice for use in the experimental model of allergic asthma wereobtained from Harlan UK, and BALB/c mice for use in the experimentalmodel of IBD were obtained from Charles River. Mice were maintained inthe SABU/CBS/Ares-MRC or National Heart and Lung Institute facilities inspecific pathogen free environments. All animal experiments outlined inthis report were undertaken with the approval of the UK Home Office.

Sensitisation and Allergen Exposure

For the experimental model of allergic asthma, BALB/c mice wild-typemice or IL-17BR knock-out mice on a BALB/c background were sensitised byintraperitoneal administration of OVA (20 μg/injection) complexed withalum, or 1:1 PBS:alum (controls), at days 0 and 12. Aerosoladministration of PBS or 1% OVA was undertaken on days 19, 20, 21 for 20minutes per day. On day 22 the animals were sacrificed and tissuescollected.

For the experimental model of IBD, BALB/c mice were sensitized by skinapplication of a 4% (w/v) solution of oxazolone (OXA) in 100% ethanol orethanol alone (controls), at day 0. Intra-rectal administration of a 3%(w/v) solution of oxazolone in 50% ethanol or 50% ethanol alone(controls) was performed at day 7. On day 9 the animals were sacrificedand tissues collected.

Administration of Anti-IL-17BR Antibodies

In the experimental model of allergic asthma, for mice receivingantibody treatment, an intraperitoneal injection of 250 μg D9.2 oranti-c-myc mouse IgG1 control antibody (clone 9E10.2) in PBS was giventwo hrs prior to each nebulisation. Each mouse received three doses ofantibody.

In the experimental model of IBD, for mice receiving antibody treatment,an intraperitoneal injection of 500 μg of D9.2 or anti-KLH mouse IgG1control antibody in PBS was given 24 hours prior to sensitization (day−1) and challenge (day 6). Thus each mouse received two doses ofantibody.

Assessing AHR

For FIG. 8( e), mice were sensitised by intraperitoneal (i.p.)administration of endotoxin-low ovalbumin and challenged daily for 6days by nebulisation with aerosolised PBS (control) or OVA.

Mice receiving D9.2 antibody treatment were given an intraperitonealinjection of 250 μg D9.2 or anti-c-myc mouse IgG1 control antibody(clone 9E10.2) in PBS 2 hours prior to each of the last 3 nebulisations.24 hours after the final aerosol challenge AHR was assessed using arestrained whole body plethysmograph (EMMS, UK). Animals wereanaesthetised, tracheostomised, and ventilated (MiniVent 845 ventilator,EMMS, UK) at a rate of 175 breaths/min, with a tidal volume of 200μl/stroke. After recording stable baseline pulmonary resistance for 3mins increasing concentrations of acetyl-β-methylcholine chloride(methacholine) (Sigma-Aldrich) were administered by aerosol for 10 secwith an ultrasonic nebuliser, and pulmonary resistance was recorded fora 3 min period. eDaq software was used to analyse airways resistance,compliance, and standard pulmonary parameters.

Mediastinal Lymph Node Restimulations

Mediastinal lymph nodes from PBS- or OVA-treated mice were pushedthrough 70 μm cell-strainers to achieve a single cell suspension. Cellswere counted and plated at 3×10⁵ cells/well on round-bottomed 96-wellplates. Cells were cultured for 72 hrs in RPMI 10% FCS alone or in thepresence of 100 μg/ml OVA. Supernatants were then collected and assayedfor IL-13 concentration using a Quantikine ELISA kit (R&D Systems).

Assessing IBD

For FIG. 9, mice were sensitised by skin application of a solution ofoxazolone in ethanol at day 0, and challenged with a solution ofoxazolone in ethanol at day 7. Controls received ethanol alone. Micereceiving antibody treatment were given an intraperitoneal injection ofD9.2 or anti-KLH mouse IgG1 antibody (control) at day −1 and day 6. Atday 9, animals were sacrificed and tissues collected. (In FIGS. 9( a)and 9(b), days 7, 8 and 9 are numbered as days 0, 1 and 2 respectively.)

At days 7, 8 and 9, mice were weighed and their general appearance andbehaviour assessed in order to assign a clinical score from 0 to 3 foreach animal, based on the method described in Wang et al., 2004, theclinical score at day 7 being set at 0 (FIG. 9 b). At day 9, mice weresacrificed and the colon of each mouse recovered, inspected and measured(FIG. 9( c)).

Example 1 Generation of Antibodies Against IL-17BR

We initially attempted to generate antibodies by immunising mice withsynthetic peptides derived from the amino acid sequence of humanIL-17BR. Despite generating monoclonal anti-peptide antibodies we failedto produce antibodies that would recognise the mature human IL-17BRprotein. We then attempted to generate antibodies against a fusionprotein of the human IL-17BR protein by immunising wild-type mice.Despite multiple immunisations and hybridoma fusions, we failed togenerate high affinity antibodies against IL-17BR.

Mice also express a form of IL-17BR and we hypothesised whether ourinability to raise a useful antibody was constrained by the lack ofnovel epitopes between the mouse and human IL-17BR molecules. Wegenerated an IL-17BR-deficient mouse line that would no longer expressIL-17BR. The IL-17BR-deficient mice were designed to remove all forms ofIL-17BR including alternatively spliced variants. The high degree ofconservation of the binding interface between human and mouse IL-17BRmay reduce the likelihood of raising blocking antibodies in wild-typemice, so removing endogenous IL-17BR may facilitate the development ofantibodies against the ligand binding site of the IL-17BR. This strategyalso increased the possibility of raising an antibody to IL-17BR thatwould block the binding of both mouse and human IL-25. This is usefulsince cross-reactive antibodies can be tested for efficacy in mousemodels of disease.

After generation and characterisation of IL-17BR-deficient animals,immunisation with IL-17BR-Fc fusion protein was performed. This strategydid prove more successful than using wild-type mice but still requiredthe screening of large numbers of hybridomas in order to identifycandidate antibodies.

A large panel of antibodies, generated in il17br^(−/−) mice immunizedagainst mouse IL-17BR-Fc fusion protein, was screened for binding tohuman and mouse IL-17BR by ELISA. One of the anti-IL-17BR antibodiesidentified (D9.2) bound well to both murine and human IL-17BR by ELISA(FIG. 1) as well as to both native mouse and human IL-17BR proteinexpressed on COS cells transfected with mouse IL-17BR cDNA or humanIL-17BR cDNA (FIG. 2).

Example 2 In Vitro Testing of D9.2

The specificity of D9.2 was tested by assaying the interaction of D9.2with other IL-17 receptor family members. D9.2 did not cross-react withIL-17A receptor (IL-17RA), IL-17C receptor (IL-17RC) or IL-17D receptor(IL-17RD)(FIG. 3).

The screen identified several antibodies which bound IL-17RB, howeveronly D9.2 was able to inhibit the interaction between human IL-25 andhuman IL-17BR (FIG. 4).

D9.2 was tested for its ability to inhibit the biological activity ofIL-25. IL-25 induces the release of type-2 cytokines, such as IL-13,initially from innate non-B/non-T (NBNT) cells (Fallon et al., 2006;Fort et al., 2001) and from T cells (Angkasekwinai et al., 2007).Furthermore, both human TK-10 (a renal carcinoma cell line) and mouserenal carcinoma (RENCA) cell lines secrete the chemokine IL-8 (known asKC in the mouse) in response to stimulation with TNF-α and IL-25 (Sayerset al., 1990).

In an in vitro bioassay, D9.2 inhibited the bioactivity triggered byIL-17BR/IL-25 binding—i.e. IL-25-dependent production of IL-13 byprimary mouse NBNT cells (FIG. 5 a) and CD4+ T/NKT cells (FIG. 5 b).

Furthermore, D9.2 inhibited KC production from IL-25-stimulated mouseRENCA cells in vitro (FIG. 6).

Significantly D9.2 was also able to inhibit the biological activity ofhuman IL-25. D9.2 inhibited IL-25-dependent IL-8 secretion by humanTK-10 cells in a dose-dependent fashion (FIG. 7).

The combination of these properties was investigated further in in vivosystems to demonstrate usefulness in the treatment of asthma. Additionalexperiments demonstrate efficacy in the treatment of IBD.

Example 3 Experimental Model of Allergic Asthma

BALB/c mice were first sensitized with the antigen OVA, before beingchallenged with aerosolised OVA. Sensitised and challenged BALB/c micedevelop a distinctive asthma phenotype. This is characterised byincreased AHR following exposure to the provocative agent methacholine,eosinophil infiltration of the airways, goblet cell hyperplasia andserum IgE secretion, as compared to control BALB/c mice challenged withPBS.

Using this model, BALB/c mice were treated at the challenge phase witheither isotype control anti-c-myc IgG1 (clone 9E10.2) or anti-IL-17BRclone D9.2. Significantly, administration of D9.2 reduced the levels ofIL-13 produced following antigen challenge and IL-5 produced followingantigen restimulation to levels similar to those found in the absence ofantigen challenge or in IL-17BR-deficient mice (FIGS. 8 a and b).Numbers of IL-13-producing cells in the lungs of antigen challenged micewere also reduced to such levels (FIG. 8 c). Similarly, disease-relatedexpansion of gamma/delta T cells was also found to be blocked followingtreatment with D9.2 (FIG. 8 d). Jin et al. (2007, J. Immunol.) haveshown that the absence of gamma/delta T cells leads to an inability todevelop AHR, suggesting that anti-IL-17BR antibody is able to inhibittwo pathways known to be essential in the development of asthma—IL-13production and gamma/delta T cell responsiveness. Furthermore, treatmentwith D9.2 reduced airways hyperreactivity, a key feature of humanasthma, in a mouse model of asthma.

Example 4 Experimental Model of Inflammatory Bowel Disease (IBD)

BALB/c mice were first sensitized with the hapten oxazolone (OXA),before being challenged by intra-rectal injection of the same chemical.Sensitized and challenged mice develop a distinctive IBD phenotype,characterised by weight loss, shortening of the colon and inflammationin the large intestine, accompanied by blood in the stools (as comparedto ethanol only controls).

Using this model, BALB/c mice were treated prior to both sensitizationand challenge with OXA with either isotype control anti-KLH IgG1 oranti-IL17BR (clone D9.2). Administration of D9.2 reduced the diseaseindex of the animals, resulting in a lower mortality rate (FIG. 9( a))and improved clinical score, i.e. reduced clinical signs of IBDmanifested in weight loss and the behaviour and appearance of theanimals (FIG. 9( b)). Furthermore, D9.2 protected against the colonshortening that results from inflammation and haemorrhage (FIG. 9( c)),and the colons of mice treated with D9.2 showed less inflammation andhaemorrhage in comparison with mice receiving anti-KLH control antibody.

Example 5 Cloning and Sequencing D9.2

To clone the immunoglobulin sequence from D9.2, RNA was isolated fromthe D9.2 cell clone and cDNA prepared by a reverse transcriptionreaction.

The immunoglobulin heavy chain (IgH) cDNA was amplified by PCR using aconserved 5′ VH region primer, MHV2 (SEQ ID NO:11) in combination anIgG1 constant region primer MHCG1 (SEQ ID NO:12).

Similarly, immunoglobulin light chain (IgK) was amplified using aconserved 5′ IgK region primers MKV3 (SEQ ID NO:13) in combination withthe kappa constant region primer MKC (SEQ ID NO:14).

The thermostable polymerase Phusion (NEB F-531L) was used throughout forPCR reactions.

The D9.2 amplification products of VH2+MHCG1 were directly ligated intothe pCRII®Blunt-TOPO® vector using the TOPO-blunt Cloning® kit (Cat45-0245), as were the amplification products of the light chainamplification reaction. E. coli TOP10 bacteria transformed with theligated pCRII-blunt vector constructs were cloned on LB-ampicillin-XGalagar plates, by picking white colonies onto an agar grid and into thePCR screening mixture. The cloned plasmid inserts were PCR-amplified.The amplification products were gel electrophoresed and the predictedproducts identified. Overnight cultures (5 ml) of each clone, producingthe correct-sized PCR amplification product, were processed using theQIAprep Spin Miniprep Kit Protocol (cat 27106), to produce DNA plasmidminipreps. Each selected plasmid was sequenced in both directions usingM13 forward and reverse primers.

The complete cycle of RT-PCR, cloning, and DNA sequence analysis wasrepeated to obtain two completely independent sets of sequenceinformation for each immunoglobulin chain.

The complete deduced nucleotide sequence of the VH and Vkappa genes areshown as SEQ ID NO:1 and SEQ ID NO:3 respectively. These sequencesinclude the leader sequences at the beginning of each variable genesegment which encodes a signal sequence which is used to transport thenewly synthesized antibody chains into the endoplasmic reticulum; theyare not present in the final heavy and light chains.

Immunology and Molecular Biology Reagents

UK Catalog Lot Article Supplier Number Numbers 10β competent NEB C3019HE. coli cells Agarose Invitrogen 15510-027 3048948 (UltraPure ™) Albuminbovine (BSA) Sigma A7030 086K1230 Ampicillin Sigma A-9518 63H0992 IL-25(murine) R&D Systems IL-25 (human) R&D Systems Oligonucleotides Sigman.a. Oxazolone Sigma E0753 PBS Tablets Sigma P4417 017K8212 QIAprep SpinMiniprep Qiagen 27106 127150290 Kit Quantikine Murine IL-13 R&D SystemsM1300CB ELISA Kit Quick Ligation Kit NEB M2200s QuikChange ® II XL Site-Stratagene 200522-5 0870486 Directed Mutagenesis Kitstreptavidin-labelled Invitrogen dynabeads SYBR Safe DNA gel stainInvitrogen 33102 55081A TOPO-blunt cloning ® kit Invitrogen 45-02451311906 X-Gal Promega V394A 20965701 rhIL-17RD (Sef) R&D systems 2275-ILNBR015031 rmIL-17RD (Sef) R&D systems 2276-ML NAF014111 rhIL-17RC R&Dsystems 2269-IL NCJ0208081 rmIL-17RC R&D systems 2270-ML rhIL-17BR-FcR&D systems 1207-BR rmIL-17BR-Fc R&D systems 1040-BR rhIL-17RA-Fc R&Dsystems 177-IR rmIL-17RA-Fc R&D systems 4481-MR Mouse CXCL1/KC R&Dsystems MKC00B Quantikine ELISA Kit Human CXCL8/IL-8 R&D systems D8000CQuantikine ELISA Kit

ABBREVIATIONS

-   AHR Airways hyperreactivity-   ° C. Centigrade-   bp Base pairs-   CD Crohn's disease-   CDR Complementarity determining region-   DMEM Dulbecco's Modified Eagles Medium-   DNA Deoxyribonucleic acid-   ELISA Enzyme linked immuno-adsorbent assay-   FACS Fluorescence activated cell sorting-   FCS Foetal calf serum-   g Grams-   hr Hour-   HRP Horseradish peroxidase-   IBD Inflammatory bowel disease-   Ig Immunoglobulin-   i.p. intraperitoneal-   KLH Keyhole Limpet Hemocyanin-   mAb Monoclonal antibody-   min Minute-   NBNT Non B/Non T cells isolated from mouse mesenteric lymph nodes-   nm Nanometre-   OD Optical density-   OVA Ovalbumin-   OXA Oxazolone-   PBS Phosphate buffered saline-   PCR Polymerase chain reaction-   RENCA renal carcinoma-   RH Recombinant heavy chain-   RK Recombinant kappa chain-   TMB 3,3′,5,5′ tetramethylbenzidine-   UC Ulcerative colitis-   VH Immunoglobulin heavy chain variable region-   VL Immunoglobulin light chain variable region-   VK Immunoglobulin kappa light chain variable region

Sequences SEQ ID NO: 1 D9.2 VH encoding nucleotide sequencecttcttcttagcaacacctacatgtgtccactcccaggtccaattgcagcagcctggggctgagctggtgaggcctggggcttcagtgaagctgtcctgcaagacttctggctacacgttcatcagttattggatgaactgggttaagcaggggcctgagcaaggccttgagtggattggaagaattgatccttacgatagtgaaattcagtacaatcaaaagttcaaggacaaggccatattgactgtagacaaatcctccagcgcagcctacatgcaactcatcagcctgacatctgaggactctgcggtctattactgtgcaagatcggggggtttcgactggtttgcgtactggggccaagggactctggtcactgtctctgcagccaaaacgacacccccatcagtctatccactgaagggcgaattccagcacactggcggccgttacSEQ ID NO: 2 D9.2 VH amino acid sequenceFFLATPTCVHSQVQLQQPGAELVRPGASVKLSCKTSGYTFISYWMNWVKQGGPEQGLEWIGRIDPYDSEIQYNQKFKDKAILTVDKSSSAAYMQLISLTSEDSAVYYCARSGGFDWFAYWGQGTLVTVSSEQ ID NO: 3 D9.2 VL encoding nucleotide sequenceatgagtgtgctcactcaggtcctggcgttgctgctgctgtggcttacagatgccagatgtgacatccagatgactcagtctccagcctccctatctgtatctgtgggagaaactgtcaccatcacatgtcgagcaagtgagaatattaacagtaatttagcatggtatcagcagaaaaagggaaaatctcctcagctcctggtctatgatgtaacaaacttagcagatggtgtgccatcaaggttcagtggcagtggatcaggcacacaatattccctcaagatcaacagcctgcagtctgaagattttgggagttattactgtcaacatttttggcgtcctccgtacacgttcggaggggggaccaatctggaaataaaaSEQ ID NO: 4 D9.2 VL amino acid sequenceMSVLTQVLALLLLWLTDARCDIQMTQSPASLSVSVGETVTITCRASENINSNLAWYQQKKGKSPQLLVYDVTNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWRPPYTFGGGTNLEIKSEQ ID NO: 5 D9.2 VH CDR1 amino acid sequence SYWMNSEQ ID NO: 6 D9.2 VH CDR2 amino acid sequence RIDPYDSEIQYNQKFKDSEQ ID NO: 7 D9.2 VH CDR3 amino acid sequence SGGFDWFAYSEQ ID NO: 8 D9.2 VL CDR1 amino acid sequence RASENINSNLASEQ ID NO: 9 D9.2 VL CDR2 amino acid sequence DVTNLADSEQ ID NO: 10 D9.2 VL CDR3 amino acid sequence QHFWRPPYTSEQ ID NO: 11 MHV2 primer sequence atgggatggagctrtatcatsytctt (r =a/g, s = c/g, y = t/c) SEQ ID NO: 12 MHCG1 primer sequencecagtggatagacagatggggg SEQ ID NO: 13 MKV3 primer sequenceatgagtgtgctcactcaggtcctggsgttg SEQ ID NO: 14 MKC primer sequenceactggatggtgggaagatgg

REFERENCES

-   1. Angkasekwinai, P., et al., J Exp Med 204, 1509-1517 (2007)-   2. Ballantyne, S. J., et al., J Allergy Clin Immunol (2007)-   3. Fallon, P. G., et al., J Exp Med 203, 1105-1116 (2006).-   4. Fort, M. M., et al., Immunity 15, 985-995 (2001).-   5. Lajoie-Kadoch, S., Am J Physiol Lung Cell Mol Physiol 290,    L1238-46.-   6. Lee, J., et al., J Biol Chem 276, 1660-1664 (2001).-   7. Moseley, T. A., et al., Cytokine Growth Factor Rev 14, 155-174    (2003).-   8. Owyang, A. M., et al., (2006) J Exp Med 203, 843-849.-   9. Pan, G., et al., J Immunol 167, 6559-6567 (2001).-   10. Shi, Y., et al., J Biol Chem 275, 19167-19176 (2000).-   11. Tian, E., et al., Oncogene 19, 2098-2109 (2000).-   12. Wang, Y. H., et al., J Exp Med 204, 1837-1847 (2007).-   13. Rickel E. A., et al., J Immunol 181, 4299-4310 (2008).-   14. Sayers T. J., et al., Cancer Res 50, 5414-5420 (1990).-   15. Jin N., et al., J Immunol 179, 2961-2968 (2007).-   16. Heller, F., et al., Immunity 17:629-638 (2002).-   17. Fichtner-Feigl, S., et al., Mucosal Immunology 1 Suppl 1:S24-27    (2008).-   18. Buning, C., et al. Eur J Immunogenet 30:329-333 (2003).-   19. Hanauer, S. B., Alimentary pharmacology & therapeutics 27 Suppl    1:15-21 (2008).-   20. Wang, X., et al., Chinese Journal of Digestive Diseases 5,    165-168 (2004)

1.-22. (canceled)
 23. A method for the treatment or prevention ofinflammatory bowel disease, said method comprising administering to asubject in need of treatment an effective amount of an antibody moleculewhich binds IL-17BR and which comprises an antibody VH domain comprisinga VH CDR3 having an amino acid sequence substantially as set out in SEQID NO:7, optionally in a pharmaceutically acceptable carrier.
 24. Themethod of claim 23, wherein the inflammatory bowel disease is ulcerativecolitis or Crohn's disease. 25.-30. (canceled)
 31. The method of claim23, wherein the VH domain further comprises a CDR1 having an amino acidsequence substantially as set out in SEQ ID NO:5 and a CDR2 having anamino acid sequence substantially as set out in SEQ ID NO:6.
 32. Themethod of claim 23, wherein the VH domain comprises a human frameworkregion.
 33. The method of claim 23, wherein the VH domain comprises SEQID NO:2.
 34. The method of claim 23, wherein the antibody furthercomprises a VL domain with a CDR1 having an amino acid sequencesubstantially as set out in SEQ ID NO:8, a CDR2 having an amino acidsequence substantially as set out in SEQ ID NO:9 and a CDR3 having anamino acid sequence substantially as set out in SEQ ID NO:10.
 35. Themethod of claim 23, wherein the VL domain comprises a human frameworkregion.
 36. The method of claim 23, wherein the VL domain comprises SEQID NO:
 4. 37. The method of claim 23, wherein the antibody comprises aVH domain comprising a CDR1 having the amino acid sequence of SEQ IDNO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3having the amino acid sequence of SEQ ID NO:7; and a VL domaincomprising a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2having the amino acid sequence of SEQ ID NO:9, and a CDR3 having theamino acid sequence of SEQ ID NO:10.
 38. The method of claim 23, whereinthe antibody molecule is a Fab, F(ab′)₂, or single chain variablefragment (scFv) antibody fragment.
 39. The method of claim 23, whereinthe antibody molecule comprises an antibody constant region.
 40. Themethod of claim 39, wherein the constant region is an IgG1 or IgG4constant region.
 41. The method of claim 39, wherein the antibodymolecule comprises a whole antibody.